Bovine Hemoglobin Solution Research Articles

(43) - A Newly Produced Polymerized Bovine Hemoglobin and Hemoglobin-Based Heart Preservation Solution Prolonged Preservation Duration of Donor Heart in Cold Static Storage and Reduced Myocardial Injury After Allograft Transplantation

(43) - A Newly Produced Polymerized Bovine Hemoglobin and Hemoglobin-Based Heart Preservation Solution Prolonged Preservation Duration of Donor Heart in Cold Static Storage and Reduced Myocardial Injury After Allograft Transplantation

Influence of bovine hemoglobin on the disruption of fluorescence resonance energy transfer between zinc sulfide quantum dots and fluorescent silica nanoparticles

Herein, the fluorescence resonance energy transfer (FRET) system composed of Zinc sulfide quantum dots (ZnS QDs) and fluorescent silica nanoparticles (FS) and the influence of bovine hemoglobin (BHb) on the system were studied using spectroscopy. UV–vis, SEM and XRD were used to characterize ZnS QDs. FT-IR spectroscopy, Zeta potential, DLS and fluorescence spectroscopy confirmed the synthesis of FS. The mechanism and efficiency of FRET in aqueous solution and BHb solution has been analyzed by fluorescence spectroscopy and Förster's non-radiative energy transfer theory, which implied that in the absence of BHb, there is dynamic quenching between ZnS QDs and FS. In the presence of BHb, it is static quenching. And BHb interfered with the stability of the ZnS QDs-FS FRET system and reduced FRET efficiency. Moreover, steady-state, synchronous fluorescence spectra and Raman spectroscopy showed that ZnS QDs caused an increase of hydrophilicity of the chromophore microenvironment, and FS may induce heme degradation to form fluorescent heme product, resulting in increased fluorescence intensity. This investigation will be significant in exploring the toxicity profile of ZnS QDs-FS FRET system for their in vivo.

Ex-Vivo Normothermic Limb Perfusion With a Hemoglobin-Based Oxygen Carrier Perfusate.

Ex-vivo normothermic limb perfusion (EVNLP) has been proven to preserve limb viability better than standard cold storage. Perfusates containing packed red blood cells (pRBC) improve outcomes when compared to acellular perfusates. Limitations of pRBC-based perfusion include limited availability, need for cross match, mechanical hemolysis, and activation of pro-inflammatory proteins. Hemoglobin-based oxygen carrier (HBOC)-201 (Hemopure) is a solution of polymerized bovine hemoglobin, characterized by low immunogenicity, no risk of hemolytic reaction, and enhanced convective and diffusive oxygen delivery. This is a preliminary study on the feasibility of EVNLP using HBOC-201 as an oxygen carrier. Three porcine forelimb perfusions were performed using an established EVNLP model and an HBOC-201-based perfusate. The perfusion circuit included a roller pump, oxygenator, heat exchanger, and reservoir. Electrolytes, limb temperature, weight, compartment pressure, nerve conduction, and perfusion indicated by indocyanine green angiography and infra-red thermography were monitored. Histological evaluation was performed with hematoxylin and eosin and electron microscopy. Three limbs were perfused for 21.3±2.1hours. Muscle contractility was preserved for 10.6±2.4hours. Better preservation of the mitochondrial ultrastructure was evident at 12hours in contrast to crystallization and destruction features in the cold-storage controls. An HBOC-201-EVNLP produced outcomes similar to RBC-EVNLP with preservation of muscle contractility and mitochondrial structure.

Cr3+‐Doped Broadband NIR Garnet Phosphor with Enhanced Luminescence and its Application in NIR Spectroscopy

AbstractBroadband near‐infrared (NIR) phosphor‐converted light emitting diode (pc‐LED) is demanded for wearable biosensing devices, but it suffers from low efficiency and low radiance. This study reports a broadband NIR Ca3‐xLuxHf2Al2+xSi1−xO12:Cr3+ garnet phosphor with emission intensity enhanced by 81.5 times. Chemical unit co‐substitution of [Lu3+−Al3+] for [Ca2+−Si4+] is responsible for the luminescence enhancement and further alters the crystal structure and electronic properties of the garnet. Using the optimized phosphor, a NIR pc‐LED with photoelectric efficiencies of 21.28%@10 mA, 15.75%@100 mA and NIR output powers of 46.09 mW@100 mA, 54.29 mW@130 mA is fabricated. The high power NIR light is observed to penetrate upper arms (≈8 cm). For application in NIR spectroscopy, the NIR pc‐LED is used as light source to measure transmission spectra of water, alcohol, and bovine hemoglobin solution. These results indicate the NIR garnet phosphor to be a promising candidate for NIR pc‐LED.

Binding interactions of MoS2 quantum dots with hemoglobin and their adsorption isotherms and kinetics in vitro

To elucidate the binding interaction and adsorption isotherms and kinetics of molybdenum disulfides quantum dots (MoS2 QDs) with bovine hemoglobin (BHB) in vitro, it is urgent to investigate their binding mechanism in this work. The experimental results indicated that MoS2 QDs partly induced denaturation and expansion of secondary structure of BHB and the microenvironment changes of heme by the ground state complex formation of BHB with MoS2 QDs. The tertiary structure and the thermal denaturation of BHB on MoS2 QDs surface are not obviously different from only BHB solution. The experimental results were verified correctness with molecular modeling analysis. The Soret band changes indicated that the presence of MoS2 QDs partly protects the oxidation process of BHB by H2O2. In addition, the kinetics of BHB adsorption onto MoS2 QDs follows the pseudo-second-order kinetic model with high adsorption capacity, their adsorption was a fast process. This in vitro study could act as a precedent in understanding the interaction mechanism of biomolecules with MoS2-based nanomaterials.

Novel and Sensitive Core-Shell Nanoparticles Based on Surface Plasmon Resonance

In this paper, a rough silver core-shell nanoparticle with strong electric field enhancement in the vicinity of a bumpy structure on the silver core-shell surface is reported. A dipolar plasmonic mode of the silver nanoshell is investigated by using the quasi-static approach and plasmon hybridization theory, which analytical results identify the electric field enhancement spectra in which the enhancement is optimized. As the silver shell thickness is small, the hot spots play an important role in the plasmonic field enhancement. In addition, the deposition of a rough silver shell can generate a stronger near-field enhancement near the silver surface which is more desirable than that of a smooth silver shell for sensitive detection based on SPR and surface enhanced Raman scattering (SERS). The plasmonic field enhancement of a bumpy silver core-shell nanoparticle permits the detection and characterization of bovine serum albumin (BSA) protein molecule and hemoglobin solution with a high sensitivity.

Cardiac function during resuscitation from hemorrhagic shock with polymerized bovine hemoglobin-based oxygen therapeutic

Hemorrhage impairs myocardial contractile function and decreases oxygen delivery. This study investigates how polymerized bovine hemoglobin (PolyHb) solutions affect cardiac function after resuscitation from hemorrhagic shock (HS). Hamsters were hemorrhaged and resuscitated with PolyHb at 8.5 g/dL and 11.5 g/dL. Left ventricle (LV) function was assessed during shock and resuscitation using a miniaturize conductance catheter. PolyHb resuscitation had no beneficial effects in cardiac function; it increased cardiac afterload and systemic vascular resistance (SVR) of 46 and 116% for 8.5 and 11.5 g/dL, respectively. Study findings indicate that preclinical evaluation of cardiac function is essential to develop safe and efficacious alternatives to blood transfusion.

Specific detection of avidin–biotin binding using liquid crystal droplets

Poly(acrylicacid-b-4-cynobiphenyl-4′-undecylacrylate) (PAA-b-LCP)-functionalized 4-cyano-4′-pentylbiphenyl (5CB) droplets were made by using microfluidic technique. The PAA chains on the 5CB droplets, were biotinylated, and used to specifically detect avidin–biotin binding at the 5CB/aqueous interface. The avidin–biotin binding was characterized by the configurational change (from radial to bipolar) of the 5CB droplets, as observed through a polarized optical microscope. The maximum biotinylation was obtained by injecting a >100μg/mL biotin aqueous solution, which enabled a limit of detection of 0.5μg/mL avidin. This droplet biosensor could specifically detect avidin against other proteins such as bovine serum albumin, lysozyme, hemoglobin, and chymotrypsinogen solutions. Avidin detection with 5CBPAA-biotin droplets having high sensitivity, specificity, and stability demonstrates new applications of the functionalized liquid crystal droplets that can detect specific proteins or other analytes through a ligand/receptor model.

Platelet Aggregation Increased by Advanced Glycated Hemoglobin

Introduction: Many studies have provided strong evidence for an association between diabetes complications and an increase in platelet (PLT) reactivity. Though some metabolic abnormalities have been reported as the major causes of this reactivity and malfunction, the precise mechanism has not been fully elucidated. Aim: The aim of this study is to investigate the effect of advanced glycation end product of hemoglubin (AGE-Hb) on human PLT reactivity and malfunction. Materials and Methods: A solution of bovine hemoglobin was prepared with fructose in phosphate buffer. The solution was then sterilized and incubated under sterile conditions at 37°C in the dark. The control solution was prepared in the same way, but without fructose. Human PLTs were isolated and prepared from 15 healthy volunteers, men and women. Blood was collected in the morning from fasting healthy subjects using a 21-gauge needle with 117 mM sodium citrate (1:9 v/v), as an anticoagulant. Fluorescence measurements were performed using a Hitachi F-4500 spectrofluorometer. PLT aggregation was measured using the photometric system Packs-4 aggregometer. Results: The relative fluorescence intensity increased in Hb samples but not in controls that incubated with fructose. PLTS aggregation did not change in controls while after incubation with 10, 22, 30-days fructose-glycated Hb decreased by 10, 12, 30%, respectively. Taken together our data show that PLT secondary phase of ADP-induced aggregation gradually affected with advanced increase in Hb glycation.

Investigation of the effects of a polymerised bovine haemoglobin solution on tension in isolated canine saphenous artery.

To investigate the vasoconstriction induced by a polymerised bovine haemoglobin solution, Hb-200, in isolated canine arteries. Rings of canine saphenous artery, from euthanatized dogs, were mounted between stainless steel wires in Krebs' solution (95% O2 , 5% CO2 , 37°C) for isometric tension recording. Following incubation with Hb-200, cumulative concentration response curves to phenylephrine (vasoconstrictor) and acetylcholine (vasodilator) were investigated. Responses to acute addition of Hb-200 were also examined in pre-constricted or pre-dilated arteries. Responses were further studied in the presence or absence of the endothelium, inhibitors of endothelium-dependent vasodilation (L-NAME, charybdotoxin and apamin), an endothelin antagonist (BQ-788) and the antioxidant superoxide dismutase. Incubation with Hb-200 (0·2 or 2 g/L) significantly enhanced phenylephrine-induced contraction (decreasing half maximal effective concentration, EC50 , P=0·0035) and inhibited acetylcholine-induced relaxation (increasing EC50 , P<0·0001). Acute addition of Hb-200 (0·2 or 2 g/L) significantly increased tension in pre-constricted arteries (P=0·0059) and reversed relaxation in pre-dilated arteries (P=0·0005). These acute responses were abolished in endothelium-denuded arteries and arteries incubated with L-NAME. Responses to Hb-200 were unaffected by incubation with charybdotoxin and apamin, BQ-788, or superoxide dismutase. Low concentrations of Hb-200 enhance vasoconstriction in isolated canine saphenous artery, primarily by antagonism of nitric oxide. This effect may be detrimental in some dogs (e.g. those at risk of volume overload) but beneficial in others (e.g. those in septic shock).

Aqueous synthesis and characterization of bovine hemoglobin-conjugated cadmium sulfide nanocrystals

Cadmium sulfide (CdS) nanocrystals with average diameter about 5.5nm were synthesized in aqueous solution of bovine hemoglobin (BHb) via simple biomimetic method. Powder X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM) and selected area electron diffraction (SAED) characterizations were used to determine the structure and morphology of CdS nanocrystals. It was revealed that amount of BHb, chelating of Cd2+ to BHb and reaction temperature were key factors in controlling shape and dispersion of CdS nanocrystals. The binding sites of BHb to CdS nanocrystals and the change of secondary structure of protein have been identified by Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy. It was found that conjugating of BHb with Cd2+ and CdS could protect nanocrystals from agglomerating. Moreover, the thermostability of BHb enhanced after conjugating with CdS nanocrystals. The interaction mechanism of BHb with Cd2+/CdS was also proposed. The quantum-confined effect of CdS nanocrystals was confirmed by ultraviolet–visible (UV–vis) spectrum. The nanocrystals exhibited a well-defined photoluminescence (PL) emission feature at about 510nm with narrow full width at half maximum (FWHM).

Investigation on the interaction of the toxicant, gentian violet, with bovine hemoglobin

Gentian violet (GV) is a well-known triarylmethane dye that is used in aquacultural, industrial and medicinal fields. But concerns in growing number have been paid to its potential health problems to human beings and its hazardous effects to environment. Herein, the toxic interaction of GV with bovine hemoglobin (BHb) was investigated by a series of spectroscopic methods and molecular modeling method. The fluorescence emission profile exhibited a remarkable quenching upon addition of GV to the buffered aqueous solution of BHb and the analysis of results revealed the dominant role of static quenching mechanism in GV-BHb interaction. The negative ΔH and positive ΔS values demonstrated that the electrostatic interactions mainly stabilized this toxicantprotein complex. Synchronous fluorescence, UV-Vis absorption and CD spectroscopic studies proved that the conformational change of BHb was induced by GV's combination. Molecular modeling studies exhibited the binding mode of GV-BHb complex and the detailed information of related driving forces. During the (1)H nuclear magnetic resonance spectra ((1)H NMR) study, the chemical shift perturbation and spin-lattice relaxation times of different protons were further used to investigate the interaction of GV with BHb and the results indicated that GV bound orientationally to BHb.

Ultraviolet Irradiation-Dependent Fluorescence Enhancement of Hemoglobin Catalyzed by Reactive Oxygen Species

Ultraviolet (UV) light has a potent effect on biological organisms. Hemoglobin, an oxygen-transport protein, plays an irreplaceable role in sustaining life of all vertebrates. In this study we scrutinize the effects of ultraviolet irradiation (UVI) as well as visible irradiation on the fluorescence characteristics of bovine hemoglobin (BHb) in vitro. Data show that UVI results in fluorescence enhancement of BHb in a dose-dependant manner. Furthermore, UVI-induced fluorescence enhancement is significantly increased when BHb is pretreated with hydrogen peroxide (H2O2), a type of reactive oxygen species (ROS). Meanwhile, The water-soluble antioxidant vitamin C suppresses this UVI-induced fluorescence enhancement. In contrast, green light irradiation does not lead to fluorescence enhancement of BHb no matter whether H2O2 is acting on the BHb solution or not. Taken together, these results indicate that catalysis of ROS and UVI-dependent irradiation play two key roles in the process of UVI-induced fluorescence enhancement of BHb.

Separation and flux characteristics in cross-flow ultrafiltration of bovine serum albumin and bovine hemoglobin solutions

The flux behavior in the separation of equimolar bovine serum albumin (BSA) and bovine hemoglobin (HB) in aqueous solutions by cross-flow ultrafiltration (UF) was investigated, in which polyacylonitrile membrane with a molecular weight cut-off (MWCO) of 100 kDa was used. BSA and HB have comparable molar mass (67,000 vs. 68,000) but different isoelectric points (4.7 vs. 7.1). The effects of process variables including solution pH (6.5, 7.1, and 7.5), total protein concentration (1.48 and 7.40 <TEX>${\mu}M$</TEX>), transmembrane pressure (69, 207, and 345 kPa), and solution ionic strength (with or without 0.01 M NaCl) on the separation were examined. It was shown that the ionic strength had a negligible effect on separation performance under the conditions studied. Although BSA and HB are not rigid bodies, the flux decline in the present cross-flow UF did not result from the mechanism of cake filtration with compression. In this regard, the specific cake resistance when pseudo steady-state was reached was evaluated and discussed.

Development of a validated process for manual preparation of dental transmission instruments

The goal of the study was to develop a validated manual preparation process that conforms to the requirements of validation guidelines. Twelve dental transmission devices from various manufacturers (turbines, handpieces, and contra-angle handpieces) were artificially contaminated with bovine hemoglobin for the test. Ten microliters (corresponding to 800 μg) of bovine hemoglobin solution (concentration 80 mg/ml) was pipetted into the spray water and spray air channels. The manual preparation was conducted by blowing air through the spray channels of the transmission instruments through an attachment to a treatment unit (model 1060T, KaVo, Biberach, Germany) for 5 s. The spray channels were cleaned with WL-Clean (Alpro, Georgen, Germany) as directed by the manufacturer. The spray channels were disinfected with WL-Cid (Alpro) and the spray channels were blow-dried with WL-Dry (Alpro) at the end of the exposure time as directed by the manufacturer. To determine the protein content (protein residue analysis) in the channels of the transmission instruments, 2 ml of an alkaline SDS solution (1%; pH 11) was flushed through the channels. For the quantitative protein residue analysis, the Biuret method was used as described in DIN EN 15883-1:2006. After the application of this method, all results of the protein residue analysis were within the acceptance criteria of the validation guideline. The newly developed manual preparation process is therefore confirmed as suitable from a hygienic viewpoint for preparation of transmission instruments in the dental practice.

Anticancer activity of phenoxazines produced by bovine erythrocytes on colon cancer cells

The present study investigated the anticancer activity of 2-aminophenoxazine-3-one (Phx-3) and 2-amino-4,4 alpha-dihydro-4 alpha,7-dimethyl-3H-phenoxazine-3-one (Phx-1), which were obtained by improved preparation methods using bovine erythrocyte suspension, on colon cancer cell lines COLO201, DLD1 and PMCO1 in vitro. The preparation methods for Phx-1 and Phx-3 had the advantages of extensively shortening reaction time and reducing sample volumes up to one-seventh during treatment, compared with the conventional method using bovine hemoglobin solution, resulting in extensive reduction of handling time. Phx-1 and Phx-3 thus obtained were identified as pure by the absorption spectra and NMR spectra. These phenoxazines exerted strong, dose-dependent anticancer activity against colon cancer cell lines COLO201, DLD1 and PMCO1 in vitro and induced apoptosis of these cells. The present results demonstrate that Phx-1 and Phx-3, which were prepared by extensively improved methods using bovine erythrocytes, may be useful as therapeutic drugs against colon cancer that is intractable to chemotherapy.

Bovine Blood and Neuromuscular Paralysis as a Bridge to Recovery in a Patent with Severe Autoimmune Hemolytic Anemia

Hemoglobin solutions have several advantages as substitutes for erythrocyte transfusions. They have a prolonged shelf life, do not require cross-matching, are associated with few transfusion reactions, and are effective in delivering oxygen to the tissues. Despite the potential clinical utility of these solutions, they have not achieved widespread use. HbOC-201 (Hemopure, Biopure, Cambridge, MA, USA) is a bovine hemoglobin solution that has been FDA approved for compassionate use in Jehovah's witness patients requiring transfusions. Here we report a case of a patient with hemoglobin H disease who developed a severe life-threatening mixed warm and cold antibody mediated autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP) who was successfully treated with HbOC-201 as an adjunct to blood transfusion.

Applicability of the exponential time dependence of flux decline during dead-end ultrafiltration of binary protein solutions

Flux decline in the dead-end ultrafiltration (UF) of binary solutions of bovine serum albumin (BSA, pI 4.7, MW 67,000) and hemoglobin (Hb, pI 7.1, MW 68,000) with polyethersulfone (PES, 100 kDa) and polyacrylonitrile (PAN, 100 kDa) membranes was studied. Key factors affecting the flux decline including solution pH (6.00–7.50), total protein concentration (1.5–9.0 μM), transmembrane pressure (TMP, 10–50 psi), and ionic strength (0.01–0.1 M) were systematically investigated. It was shown that the blocking filtration law could satisfactorily analyze the flux decline behavior. A simplified exponentially time-dependent model was adopted to describe the dynamics of flux decline during UF process, and used to determine the adequate time for membrane cleaning. The mechanism of membrane fouling was also analyzed by blocking filtration law, in which the standard blocking always dominated at the early stage in such UF processes. The fouling mechanism strongly depended on hydrophobic characteristics of the membranes, concentration and charge of the proteins, as well as pH and ionic strength of the solutions.

The effect of the polymerized bovine haemoglobin solution, Hb‐200, on endothelial function in isolated arterial rings from rats

Haemoglobin-based oxygen carriers have been developed as 'blood substitutes'. Early cross-linked haemoglobins caused marked vasoconstriction, however, polymerized haemoglobin solutions, such as the bovine product haemoglobin glutamer-200 (bovine) (Hb-200), are said to have lesser vascular effects. The aim of this study was to quantify the effect of Hb-200 on isolated rat arterial rings. The actions of Hb-200 were investigated using rings of aorta and second-order mesenteric arteries from female Wistar rats. The arterial rings were mounted for isometric tension recording. Addition of Hb-200 (1 microM) to arterial rings, precontracted with phenylephrine (50-70% maximum phenylephrine-induced tone), resulted in vasoconstriction. Addition of Hb-200 to arteries precontracted with phenylephrine and relaxed with acetylcholine (1 microM) also caused contraction. Furthermore preincubation with Hb-200 (1 microM) enhanced the response to phenylephrine (1 nm to 10 microM) and impaired acetylcholine-induced relaxation (10 nm to 10 microM). Responses to Hb-200 were similar to those in response to the nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (L-NAME, 100 microM), alone or in combination with Hb-200. These data demonstrate that Hb-200 has a significant vasoconstrictor effect similar to L-NAME. Thus the vascular actions of Hb-200 are consistent with activity as a NO scavenger. This may have implications for the clinical use of Hb-200.

Oxygen Carriers in Cardiac Surgery

SUMMARYArtificial oxygen carriers – the hemoglobin‐based oxygen carriers (HBOCs) and the perfluorocarbon (PFC) emulsions – have been developed with the aim of avoiding the risks associated with allogeneic blood transfusions. H BOCs are solutions of free human or bovine hemoglobin, modified by internal crosslinking and by polymerization so as to avoid renal toxicity and to increase the intravascular lifetime. Last‐generation PFC emulsions are formed by lipid particles enclosing a synthetic hyperfluorinated, chemically inert hydrocarbon, perflubron, which dissolves gases without binding them. In order to be efficient, the emulsions must be used with 100% oxygen.HBOCs and PFC emulsions were found to be safe and to reduce allogeneic blood transfusions in preclinical and clinical phase I/II studies. One PFC emulsion (Oxygent®) and three H BOCs (DCLHb, Hemolink® and Hemopure®) were used in phase II and III clinical trials in cardiac surgery with cardiopulmonary bypass (CPB). HBOCs were well tolerated but increased the number of adverse events, mainly hypertension, jaundice‐like syndrome and elevation of the blood levels of hepatic and pancreatic enzymes. The effects of HBOCs on transfusion avoidance were modest, limited to the first postoperative days, without decreasing the total number of transfused red blood cell (RBC) units. Oxygent® was used in combination with acute normovolemic hemodilution in phase II studies. It was safe, had modest effects on transfusion avoidance in the post‐CPB period (until 5 days for the highest administered dose), but did not decrease the number of allogeneic red blood cell units transfused per patient. A phase III clinical trial was voluntarily stopped before completion because of an increased incidence of adverse events (stroke). More evidence is warranted before oxygen carriers can be recommended for routine use in cardiac surgery.